ROLE OF FREE APOLIPOPROTEIN-A-I IN CHOLESTEROL EFFLUX - FORMATION OF PRE-ALPHA-MIGRATING HIGH-DENSITY-LIPOPROTEIN PARTICLES

This article characterizes products formed by the interaction of purif ied apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were i ncubated with different concentrations of purified apoA-I (1 to 30 mu g/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-di mensional (agarose :polyacrylamide nondenaturing gradient gel) electro phoresis. At any given concentration of apoA-I, the rate of cellular c holesterol efflux appeared linear over 24 hours. Incubating purified a poA-I with fibroblasts for 4 hours, we detected five pre-alpha lipopro teins with particle sizes between 114 and 684 kDa. Formation of pre-al pha lipoproteins was concentration-dependent. At low concentrations (b elow 5 mu g/mL apoA-I), all purified apoA-I (with pre-beta mobility) w as converted to pre-alpha lipoproteins. At higher concentrations (grea ter than 5 mu g/mL apoA-I), more apoA-I remained with pre-beta mobilit y. The pre-alpha lipoproteins were characterized by colocalization of apoA-I particles with C-14-cholesterol and P-32-phospholipids. Results showed that the pre-alpha particle of lowest molecular weight contain ed phospholipid and apoA-I but no cholesterol. The remaining pre-alpha particles contained all three substances. When pre-alpha particles we re subjected to ultracentrifugation, all particles floated at d<1.21 g /mL with some of the smallest phospholipid apoA-I only particles being present in the d>1.21 g/mL fraction. Based on these results, we postu lated that in the first stages of reverse cholesterol transport, pre-a lpha lipoproteins are formed by the interaction of lipid free apoA-I a nd peripheral cells.